The facultative intracellular bacteria, Legionella pneumophila, is an important cause of pneumonia in North America. It continues to be a significant cause of morbidity and mortality, particularly among immunocompromised patients. The ability of L. pneumophila to cause pneumonia is dependent on its capacity to survive and to multiply in a complex endosomal compartment within alveolar phagocytic cells. The role of the bacterial gene products, that are induced in response to the intracellular environment, in intracellular survival and adaptation of L. pneumophila is not known. Moreover, the bacterial immunogenic targets expressed by intracellular bacteria are poorly defined. Thus, our knowledge is still inadequate to develop effective means of prevention and therapeutic intervention. Using techniques of two dimensional SDS- PAGE of macrophage-grown L. pneumophilia, we have shown that intracellular bacteria manifest the induction in expression of 35 proteins (MI, for macrophage-induced) within the phagosome. One of the MI genes, gspA, is essential for survival and growth of L. pneumophila in phagocytic cells. We hypothesize that the MI genes of L. pneumophila are important for survival and adaptation of the bacteria in the intracellular environment of phagocytic cells. To test this hypothesis, we will clone and characterize two unique MI genes with respect to regulation of their expression and function in L. pneumophila-infected human alveolar macrophages. The first MI gene encodes for a protein specifically induced during the first 4h of infection. In contrast, the second gene encodes for a protein that is expressed only during the late stages of the infection. Since the 2D-SDS-PAGE technique suffers from many limitations including undetectable levels of protein synthesis during the initial few hours of the infection, we will test our hypothesis through the use of differential display polymerase chain reaction (DD-PCR). The MI genes will be molecularly characterized and the regulation of their expression will be evaluated at several stages of the infection. To evaluate the role of the MI genes in the cell biology of the intracellular infection, mutants of L. pneumophila will be constructed in these genes. This work will allow us to characterize the role of the MI genes in the intracellular infection, and may help in elucidation of the phagosomal "microenvironment" that Legionella reside in within the phagocyte.